In recent years, polyoxyalkylene derivatives are used in extremely large field as materials for imparting the long circulation property in the blood, the function of targeting a target site, etc. to polypeptides, enzymes, antibodies, nucleic acid compounds including genes and oligonucleic acids, nucleic acid medicines, and other physiologically active substances. This is because polyoxyalkylene derivatives show a weak interaction with other living-body components due to the steric repulsion effect. It is known that physiologically active substances modified with polyoxyalkylene derivatives or drug carriers obtained by modifying liposomes or the like with polyoxyalkylene derivatives exhibit the long circulation property in the internal blood for a longer period than the unmodified ones. It has been reported that the higher the molecular weight of the polyoxyalkylene derivatives, the higher the effect. Furthermore, by combining an active group or an antibody with an end of such a polyoxyalkylene derivative, a targeting function can also be imparted. In the field of drug delivery systems, polyoxyalkylene derivatives have become an exceedingly useful and indispensable material. Because of this, there is a desire for a polyoxyalkylene derivative which has a higher purity and a low impurity content, from the standpoints of the performance and safety of drugs to be produced using the derivative. Polyoxyalkylene derivatives having various frameworks have been developed so far, and various impurities generate as by-products depending on the methods of production. Examples thereof are shown below.
(Case A: Diol Impurity in One-End-Blocked Linear Polyoxyalkylene Derivative)
Examples thereof first include the impurities called diols that are polyoxyalkylene derivatives terminated at each of both ends by a hydroxyl group and are contained in linear polyoxyalkylene derivatives having one hydroxyl group, which are the most widely used polyoxyalkylene derivatives.
When a polyoxyalkylene derivative having one hydroxyl group is to be produced, the derivative can be obtained usually by addition-polymerizing an alkylene oxide using a corresponding alcohol as an initiator and further using an alkali catalyst. In this process, when the alkylene oxide undergoes addition polymerization with water which has come into the initiator alcohol or remains in the reaction tank or with an impurity having hydroxyl groups at both ends which has been formed by decomposition of the initiator, then the alkylene oxide addition-polymerizes with each end to produce as a by-product a diol having a molecular weight which is two times the molecular weight of the main component. Since a large amount of an alkylene oxide is addition-polymerized in producing a polyoxyalkylene derivative having a high molecular weight, the amount of the initiator becomes small for the reaction vessel. As a result, the number of moles of the water in the system becomes too large relative to the initiator and, hence, the high-molecular-weight polyoxyalkylene has an increased diol content. Furthermore, the higher the molecular weight of polyoxyalkylene derivatives, the higher the melt viscosity thereof. There are hence cases where a solvent is added in the course of the addition polymerization of an alkylene oxide to lower the viscosity, or where the catalyst is supplemented in order to heighten the rate of reaction. There also are diols which generate due to the addition polymerization of the alkylene oxide with the water contained in a slight amount in those ingredients, and such diols have various molecular weights depending on the production methods. There are even cases where diols having a molecular weight which is approximately equal to or lower than that of the main component generate.
Since diols generate even when water is present in an exceedingly slight amount as stated above, it is difficult to completely inhibit the generation of such by-products. In particular, the inhibition is more difficult in the production of high-molecular-weight polyoxyalkylene derivatives, which are considered to be effective in the field of drug carriers. Such an impurity diol is causative of drug dimerization when used for drug modification. The diols are polyether compounds like the desired products, and separation and purification are difficult. Because of this, there is a strong desire for a purification technique for diminishing the diols contained in polyoxyalkylene derivatives.
(Cases B and E: Impurity in Polyoxyalkylene Derivative of Branched Type Formed After Polymerization)
One method for producing a branched polyoxyalkylene derivative having polyoxyalkylene chains in a number which is an integer of 2 or larger is a method in which a reactive polyoxyalkylene derivative is chemically combined with a compound serving as a framework and having active-hydrogen groups.
Impurities which result as by-products from this method include the reactive polyoxyalkylene derivative, the polyoxyalkylene derivative formed through hydrolysis of the reactive polyoxyalkylene derivative and resultant return of the reactive part to a hydroxyl group, the unreacted starting material, and a reaction intermediate formed by introducing only one polyoxyalkylene chain into the starting material. Examples thereof further include the diol originally contained in the reactive polyoxyalkylene derivative. The synthesis of a branched polyoxyalkylene derivative by this method results in production of impurities having various functional groups and molecular weights, and purification is especially difficult. Such a branched polyoxyalkylene derivative modifies a physiologically active substance through few bonding sites and enables the modified substance to exhibit the same property of remaining in the blood as that modified with a linear derivative. Because of the small number of bonding sites, the branched polyoxyalkylene derivative is exceedingly effective in preventing the activity of the physiological active substance itself from decreasing. There has hence been a desire for a purification technique for removing linear polyoxyalkylene derivatives.
(Case D: Diol Impurity in Multibranched Polyoxyalkylene Derivative)
With respect to branched polyoxyalkylene derivatives having three or more hydroxyl-terminated polyoxyalkylene chains and obtained by addition-polymerizing an alkylene oxide using a polyhydric alcohol such as, for example, glycerol or diglycerol as an initiator, examples of impurities include diols derived from water contained in the initiator, as in the case of linear polyoxyalkylene derivatives having one hydroxyl group. Examples thereof further include diols derived from water contained in solvents, because some initiators which are solid or have high viscosity are dissolved in organic solvents before being used for the reaction. There also are diols which generate as by-products due to solvent addition or catalyst supplementation during the reaction, as in the case described above. Since the polyhydric alcohol as the main component has a larger number of hydroxyl groups and has a larger number of moles of the alkylene oxide added per mole, those by-product diols have a molecular weight which is equal to or lower than that of the main component. Such polyfunctional polyoxyalkylene derivatives are in use as a hemostatic agent for surgical operations, etc. in the field of medicines and as a raw material for gelling agents such as sealing agents. In case where an impurity derived from a diol comes into the polyfunctional polyoxyalkylene derivative, this means that an impurity differing in molecular weight or crosslinking site enters and a desired gel strength cannot be obtained. As incase A, water removal in the stage of synthesis is exceedingly difficult and there is a strong desire for a purification technique for diminishing diols contained in a polyoxyalkylene derivative.
(Case C: Impurity Having Unreacted Hydroxyl Group resulting from Functional Group Conversion)
The polyoxyalkylene derivatives synthesized by the methods described above are thereafter subjected to chemical modification through conversion of the terminal hydroxyl groups to various functional groups. Usually, the higher the molecular weight of the polyoxyalkylene derivative, the higher the viscosity and the lower the concentration of reactive parts in the molecule. Such a polyoxyalkylene derivative has impaired conversion and, hence, hydroxyl groups remain in a small amount. Furthermore, in the case of a polyoxyalkylene derivative having a large number of hydroxyl groups in the molecule as in case D, impurities differing in the number of unreacted hydroxyl groups generate during the chemical modification, the number of kinds of such impurities being 2 or a larger integer. From the standpoints of purity improvement and yield improvement, it has become important to diminish polyoxyalkylene derivatives having unreacted hydroxyl groups.
When unreacted hydroxyl groups thus remain, the polyoxyalkylene derivative has a reduced purity and gives a drug which also has a reduced purity. There has hence been a desire for a technique for purifying a polyoxyalkylene derivative.
Various impurities generate as by-products in the synthesis of polyoxyalkylene derivatives as described above, and various proposals have been made on methods for purifying the derivatives. In particular, with respect to the purification of a polyoxyalkylene derivative having one hydroxyl group by removing the diol therefrom, the following methods have been proposed.
(Example of Purification Method for One-End-Oh Derivative: Water Content Control During Alkylene Oxide Addition)
One method is a production method used in the addition polymerization of an alkylene oxide. As shown in patent document 1 (JP-A-11-335460) and patent document 2 (US 2006/0074200), the content of water in the system in the addition reaction of ethylene oxide using an alcohol compound as a starting material is controlled on the order of ppm, and the influence of water molecules, which are causative of the diol as an impurity having a higher molecular weight, is thereby minimized to inhibit the diol from generating. However, since a large amount of an alkylene oxide is addition-polymerized in producing a polyoxyalkylene derivative having a high molecular weight, the amount of the initiator becomes small for the reaction vessel. As a result, the number of moles of the water in the system becomes too large relative to the initiator and, hence, the high-molecular-weight polyoxyalkylene has an increased diol content. The generation of the diol as a by-product is therefore unavoidable.
(Example of Purification Method for One-End-OH Derivative: Chromatographic Purification)
Among methods proposed as techniques for removing a diol generated, there is a method in which purification and separation are implemented based on molecular weight, as in the experiment on methoxypolyethylene glycol purification through dialysis reported in non-patent document 1 (Leonard et al., Macromol. Chem., 189, 1809-1817 (1988)). However, the diol having a molecular weight equal or close to that of the main component is difficult to separate by purification through dialysis.
In the experiment on methoxypolyethylene glycol purification through column chromatography with silica gel reported in non-patent document 2 (Lapienis et al., J. Bioactive Compatible Polymers, 16, 206-220 (2001)), about tens of grams of a methoxypolyethylene glycol having a molecular weight of 5,000 or lower was subjected to removal of the diol therefrom on a laboratory level. However, the amounts of the silica gel and solvent to be used are both exceedingly large as compared with the amount of the methoxypolyethylene glycol to be purified, and production on an industrial scale is difficult. Furthermore, the compounds having an increased molecular weight have reduced polarity per molecule and are hence difficult to separate. In addition, the rate of elution varies depending on molecular weight, and affects separability. It is therefore necessary for isolating a desired compound by column chromatography that a packing material and an eluent should be selected according to the kind and content of the impurity. It is also necessary to optimize conditions for development. As described above, purification through column chromatography necessitates use of large amounts of an adsorbent and a solvent and the operation is exceedingly complicated. Purification by this method is impossible depending on the molecular weight of the desired polyoxyalkylene derivative and on the impurity contained therein.
Furthermore, patent document 3 (U.S. Pat. No. 5,298,410) and patent document 4 (JP-T-2008-514693; the term “JP-T” as used herein means a published Japanese translation of a PCT patent application) disclose a method in which the hydroxyl group of a methoxypolyethylene glycol is modified with a dimethyltrityl group, acetic ester group, or phthalic ester group to enhance a difference in polarity and the modified compound is isolated by column chromatography. However, this production method necessitates temporarily chemically modifying the terminal hydroxyl group, subjecting this compound to column purification and then to deprotection, and thereafter returning the modified group to a hydroxyl group. There are hence problems that the steps are exceedingly complicated and that impurities of a new chemical species generate due to the chemical modification. In addition, it is thought that polyethylene glycol derivatives having a higher molecular weight, in particular, have a reduced difference in terminal polarity and this makes separation difficult.
(Example of Purification Method for One-End-OH Derivative: Batch Treatment with Ion-Exchange Resin)
Meanwhile, patent document 5 (WO 2006/028745) shows an example in which a methoxypolyethylene glycol, without being chemically modified, is caused to act on an ion-exchange resin formed from a polycarboxylic acid and the diol is thereby adsorbed and removed. There is a statement therein to the effect that this technique is a purification method effective also for the high-molecular-weight compound having a molecular weight of 30,000. However, the principle of this adsorption is separation for which an interaction between ether oxygen atoms in the polyethylene glycol molecule and the carboxylic acid within the resin is utilized and which is also based on a difference in molecular weight. Because of this, the purification method is not applicable to cases where the molecular weight of the diol is equal or close to the molecular weight of the methoxypolyethylene glycol.
It can be seen from the above explanations that it is difficult to remove a high-molecular-weight diol from a high-molecular-weight polyoxyalkylene derivative having a hydroxyl group at one end and that it is technically difficult to remove diols having a molecular weight not higher than the molecular weight of the main component.
[Prior-Art Documents]
[Patent Documents]
[Patent Document 1] JP-A-11-335460
[Patent Document 2] US 2006/0074200
[Patent Document 3] U.S. Pat. No. 5,298,410
[Patent Document 4] JP-T-2008-514693
[Patent Document 5] WO 2006/028745
[Non-Patent Documents]
[Non-Patent Document 1] Leonard et al., Macromol. Chem., 189, 1809-1817 (1988)
[Non-Patent Document 2] Lapienis et al., J. Bioactive Compatible Polymers, 16, 206-220 (2001)